The advent of recombinant DNA technology has greatly accelerated the development of vaccines to control epidemic, endemic, and pandemic infectious diseases (Woodrow et al, New Generation Vaccines: The Molecular Approach, Eds., Marcel Dekker, Inc., New York, N.Y. (1989); Cryz, Vaccines and Immunotherapy, Ed., Pergamon Press, New York, N.Y. (1991); and Levine et al, Ped. Ann., 22:719-725 (1993)). In particular, this technology has enabled the growth of nucleic acid vaccines. Although the first nucleic acid vaccine was only reported in 1992, the study of nucleic acid vaccines has grown dramatically and examples of this approach have been reported in a wide array of animals using numerous antigens (Tang, D. C., et al. (1992) Nature 356:152; Fynan, E. F. et al. (1993) PNAS USA 90:11478; Donnelly, J. J. et al. (1995) Nat Med 1:583; Wang, B. et al. (1993) PNAS USA 90:4156; Davis, H. L., et al. (1993) Hum Mol Genet 2:1847; Ulmer, J. B. et al. (1993) Science 259:1745; Robinson, H. L. et al. (1993) Vaccine 11:957; Eisenbraun, M. D. et al. (1993) DNA Cell Biol 12:791; Wang, B. et al. (1994) AIDS Res Hum Retroviruses 10:S35; Coney, L. et al. (1994) Vaccine 12:1545; Sedegah, M. et al. (1994) Proc Natl Acad Sci USA 91:9866; Raz, E. et al. (1994) Proc Natl Acad Sci USA 91:9519; Xiang, Z. Q. et al. (1994) Virology 199:132) (for a more comprehensive list see www.genweb.com/ Dnavax/Biblio/articles). Early murine experiments with HIV-1 DNA vaccines produced impressive immunological responses, including neutralizing antibody responses against HIV-1 and strong CTL responses against several HIV-1 antigens (Wang, B. et al. (1993) supra; Wang, B. et al. (1994) supra; Coney, L. et al. (1994) supra; Lu, S. et al. (1995) Virology 209:147; Shiver, J. W. et al. (1995) Annals of the New York Academy of Sciences 772:198; Wahren, B. et al. (1995) Ann N Y Acad Sci 772:278). However, an initial attempt to induce protective immunity with an SIV DNA vaccine in Rhesus monkeys was disappointing (Lu, S. et al. (1996) J Virol 70:3978). In contrast, an HIV-1MN Env DNA vaccine induced measurable protection against a chimeric SIV/HIV (SHIV) challenge in vaccinated cynomologous macaques (Boyer, J. D. et al. (1996) Journal of Medical Primatology 25:242). More recently, intramuscular immunization of chimpanzees with this latter vaccine engendered protection against parenteral challenge with HIV-1SF-2 (Boyer, J. D. et al. (1997) Nature Medicine 3:526). These differences may have resulted from antigenic differences in the vaccines or differences in the potency of the challenges (Lu, S. et al. (1996) supra; Boyer, J. D. et al. (1996) supra; Boyer, J. D. et al. (1997) supra). It is noteworthy to mention that in the aforementioned chimpanzee study, DNA vaccination only induced modest humoral and cellular responses despite giving 9 doses of vaccine containing a total of 2.9 mg of DNA before challenge (Boyer, J. D. et al. (1997) supra). Thus, although these results are encouraging, the immunogenicity of HIV-1 DNA vaccines must be improved before this approach achieves practical utility in large scale vaccination programs.
The mechanism though which DNA vaccines induce immunity is not fully understood. Muscle cells express low levels of MHC class 1 and do not express detectable levels of co-stimulatory molecules B7-1 and B7-2 (review by Ertl, H. C. and Z. Q. Xiang (1996) Journal of Immunology 156:3579). While it remains conceivable that muscle cells may serve as an antigen depot (Ertl and Xiang (1996) supra), their participation in the induction of MHC class I and II responses may be secondary to other antigen presenting cells (Ertl and Xiang (1996) supra). Xiang and Ertl (Ertl and Xiang (1996) supra; Xiang, Z. and H. C. Ertl (1995) Immunity 2:129) have suggested that resident dendritic cells may be involved in the primary inductive events. They showed that co-expression of GM-CSF, a cytokine know to activate growth of dendritic cells, at the site of inoculation resulted in a more rapid response to DNA vaccine encoded antigens (Xiang and Ertl (1995) supra). In contrast, co-expression of IFN-xcex3 diminished the responses (Xiang and Ertl (1995) supra). In agreement, Manickan et al. ((1997) Journal of Leukocyte Biology 61:125) showed that immunization with dendritic cells transfected with a DNA vaccine induced elevated immune responses, compared to the identical DNA vaccine given alone. In addition, dendritic cells have been shown to express antigen following intradermal vaccination with a DNA vaccines (Raz, E. et al. (1994) supra). Although inconclusive, these data strongly suggest that dendritic cells may play a substantial role in the presentation of DNA vaccine-encode antigens.
Another new class of vaccines are bacterial vector vaccines (Curtiss, In: New Generation Vaccines: The Molecular Approach, Ed., Marcel Dekker, Inc., New York, N.Y., pages 161-188 and 269-288 (1989); and Mims et al, In: Medical Microbiology, Eds., Mosby-Year Book Europe Ltd., London (1993)). These vaccines can enter the host, either orally, intranasally or parenterally. Once gaining access to the host, the bacterial vector vaccines express an engineered prokaryotic expression cassette contained therein that encodes a foreign antigen(s). Foreign antigens can be any protein (or part of a protein) or combination thereof from a bacterial, viral, or parasitic pathogen that has vaccine properties (New Generation Vaccines: The Molecular Approach, supra; Vaccines and Immunotherapy, supra; Hilleman, Dev. Biol. Stand., 82:3-20 (1994); Formal et al, Infect. Immun. 34:746-751 (1981); Gonzalez et al, J. Infect. Dis., 169:927-931 (1994); Stevenson et al, FEMS Lett., 28:317-320 (1985); Aggarwal et al, J. Exp. Med., 172:1083-1090 (1990); Hone et al, Microbial. Path., 5:407-418 (1988); Flynn et al, Mol. Microbiol., 4:2111-2118 (1990); Walker et al, Infect. Immun., 60:4260-4268 (1992); Cardenas et al, Vacc., 11:126-135 (1993); Curtiss et al, Dev. Biol. Stand., 82:23-33 (1994); Simonet et al, Infect. Immun., 62:863-867 (1994); Charbit et al, Vacc., 11:1221-1228 (1993); Turner et al, Infect. Immun., 61:5374-5380 (1993); Schodel et al, Infect. Immun., 62:1669-1676 (1994); Schodel et al, J. Immunol., 145:4317-4321 (1990); Stabel et al, Infect. Immun., 59:2941-2947 (1991); Brown, J. Infect. Dis., 155:86-92 (1987); Doggett et al, Infect. Immun., 61:1859-1866 (1993); Brett et al, Immunol., 80:306-312 (1993); Yang et al, J. Immunol., 145:2281-2285 (1990); Gao et al, Infect. Immun., 60:3780-3789 (1992); and Chatfield et al, Bio/Technology, 10:888-892 (1992)). Delivery of the foreign antigen to the host tissue using bacterial vector vaccines results in host immune responses against the foreign antigen, which provide protection against the pathogen from which the foreign antigen originates (Mims, The Pathogenesis of Infectious Disease, Academic Press, London (1987); and New Generation Vaccines: The Molecular Approach, supra).
Of the bacterial vector vaccines, live oral Salmonella vector vaccines have been studied most extensively. There are numerous examples showing that Salmonella vectors are capable of eliciting humoral and cellular immunity against bacterial, viral and parasitic antigens (Formal et al, Infect. Immun., 34:746-751 (1981); Gonzalez et al, supra; Stevenson et al, supra; Aggarwal et al, supra; Hone et al, supra; Flynn et al, supra; Walker et al, supra; Cardenas et al, supra; Curtiss et al, supra; Simonet et al, supra; Charbit et al, supra; Turner et al, supra; Schodel et al, supra, Schodel et al (1990), supra; Stabel et al, supra; Brown, supra; Doggett et al, supra; Brett et al, supra; Yang et al, supra; Gao et al, supra; and Chatfield et al, supra). These humoral responses occur in the mucosal (Stevenson et al, supra; Cardenas et al, supra; Walker et al, supra; and Simonet et al, supra) and systemic compartments (Gonzalez et al, supra; Stevenson et al, supra; Aggarwal et al, supra; Hone et al, supra; Flynn et al, supra; Walker et al, supra; Cardenas et al, supra; Curtiss et al, supra; Simonet et al, supra; Charbit et al, supra; Turner et al, supra; Schodel et al, supra, Schodel et al (1990), supra; Stabel et al, supra; Brown, supra; Doggett et al, supra; Brett et al, supra; Yang et al, supra; Gao et al, supra; and Chatfield et al, supra). Live oral Salmonella vector vaccines also elicit T cell responses against foreign antigens (Wick et al, Infect. Immun., 62:4542-4548 (1994)). These include antigen-specific cytotoxic CD8xe2x88x92T cell responses (Gonzalez et al, supra; Aggarwal et al, supra; Flynn et al, supra; Turner et al, supra; and Gao et al, supra).
Ideally, bacterial vector vaccines are genetically defined, attenuated and well-tolerated by the recipient animal or human, and retain immunogenicity (Hone et al, Vaccine, 9:810-816 (1991); Tacket et al, Infect. Immun., 60:536-541 (1992); Hone et al, J. Clin. Invest., 90:412-420 (1992); Chatfield et al, Vaccine, 10:8-11 (1992); Tacket et al, Vaccine, 10:443-446 (1992); and Mims, supra). Recently, the number of potential bacterial vector vaccines for the delivery of prokaryotic expression cassettes has grown. They now include, but are not restricted to Yersinia enterocolitica (van Damme et al, Gastroenterol., 103:520-531 (1992)), Shigella spp. (Noriega et al, Infect. Immun., 62:5168-5172 (1994)), Vibrio cholerae (Levine et al, In: Vibrio cholerae, Molecular to Global Perspectives, Wachsmuth et al, Eds, ASM Press, Washington, D.C., pages 395-414 (1994)), Mycobacterium strain BCG (Lagranderie et al, Vaccine, 11:1283-1290 (1993); Flynn, Cell. Molec. Biol., 40(Suppl.1):31-36 (1994)), and Listeria monocytogenes (Schafer et al, J. Immunol., 149:53-59 (1992)) vector vaccines.
The commercial application of DNA delivery technology to animal cells is broad and includes, in addition to vaccine antigens, delivery of immunotherapeutic agents and therapeutic agents (Darris et al, Cancer, 74(3 Suppl.):1021-1025 (1994); Magrath, Ann. Oncol., 5(Suppl 1):67-70 (1994); Milligan et al, Ann. NY Acad. Sci., 716:228-241 (1994); Schreier, Pharma. Acta Helv., 68:145-159 (1994); Cech, Biochem. Soc. Trans., 21:229-234 (1993); Cech, Gene, 135:33-36 (1993); Long et al, FASEB J., 7:25-30 (1993); and Rosi et al, Pharm. Therap., 50:245-254 1991)).
The delivery of endogenous and foreign genes to animal tissue for gene therapy has shown significant promise in experimental animals and volunteers (Nabel, Circulation, 91:541-548 (1995); Coovert et al, Curr. Opin. Neuro., 7:463-470 (1994); Foa, Bill. Clin. Haemat., 7:421-434 (1994); Bowers et al, J. Am. Diet. Assoc., 95:53-59 (1995); Perales et al, Eur. J. Biochem., 226:255-266 (1994); Danko et al, Vacc., 12:1499-1502 (1994); Conry et al, Canc. Res., 54:1164-1168 (1994); and Smith, J. Hemat., 1:155-166 (1992)).
From the onset nucleic acid vaccine studies focused on the use of DNA vaccines (Tang et al. (1992) supra; Fynan et al. (1993) supra; Donnelly et al. (1995) supra; Wang et al. (1993) supra; Davis et al. (1993) supra; Ulmer et al. (1993) supra; Robinson et al. (1993) supra; Eisenbraun et al. (1993) supra; Wang et al. (1994) supra; Coney et al. (1994) supra; Sedegah et al. (1994) supra; Raz et al. (1994) supra; Xiang et al. (1994) supra). More recently the use of RNA vaccines has been proposed as an alternative approach to the injection of DNA based nucleic vaccines (Zhou, X. et al. (1994) Vaccine 12:1510; Conry, R. M. et al. (1995) Cancer Res 55:1397). In support, xe2x80x9cnakedxe2x80x9d RNA vaccines have proven modestly immunogenic in mice (Zhou et al. (1994) supra; Conry et al. (1995) supra). An RNA vaccine based on a recombinant Semliki Forrest Virus that expressed the SIV-PBj14 Env gene engendered protection against SIV-PBj14 (Mossman, S. P. et al. (1997) Journal of Virology 70:1953).
RNA vaccines would offer two main advantages of over DNA vaccines. First, RNA vaccines would avoid placing vaccinees at risk of an integration event, which over a human life span might lead to the development of malignancy. Second, RNA vaccines would avoid the barrier function of the nuclear membrane. This is particularly relevant given that antigen expression in non-replicating antigen presenting cells is central to the induction of immunity using nucleic acid vaccines (Ertl and Xiang (1996) supra; Xiang and Ertl (1995) supra; Manickan et al. (1997) supra).
Thus, it is desirable to have an efficient method of delivering RNA to eukaryotic cells, such as mammalian cells, such that, the RNA can be expressed in the eukaryotic cell. Furthermore, it is also desirable to have a system permitting efficient delivery of RNA molecules to mucosal tissue in addition to permitting parenteral delivery of RNA molecules.
The invention provides a system for delivery of RNA molecules to eukaryotic cells, e.g., cells of mucosal tissue. The invention is based at least in part on the discovery that bacteria which are capable of invading eukaryotic cells can deliver RNA molecules to eukaryotic cells and tissues, and where appropriate, the RNA can be translated if the RNA contains the appropriate regulatory elements.
Accordingly, in one embodiment, the invention provides an isolated bacterium comprising a DNA which is transcribed into a messenger RNA molecule in the bacterium, wherein the RNA is capable of being translated in a eukaryotic cell, or is an antisense RNA or a catalytic RNA. The DNA can be heterologous with respect to the bacterium. The DNA can be operably linked to a prokaryotic promoter, e.g., the E. coli NirB promoter. Alternatively, the DNA can be operably linked to a first promoter, and the bacterium further comprises a gene encoding a polymerase, which is capable of mediating transcription from the first promoter, wherein the gene encoding the polymerase is operably linked to a second promoter. In a preferred embodiment the second promoter is a prokaryotic promoter. In a preferred embodiment, the polymerase is a bacteriophage polymerase, e.g., T7 polymerase, and the first promoter is a bacteriophage promoter, e.g., T7 promoter. The DNA which is capable of being transcribed into said RNA and the gene encoding a polymerase can be located on one or more plasmids. However, in a preferred embodiment, the DNA is located on the bacterial chromosome.
In a preferred embodiment, the RNA can be translated in a eukaryotic cell. For allowing efficient translation in a eukaryotic cell, the RNA preferably comprises a Cap Independent Translation Enhancer (CITE) sequence. The RNA can further comprise additional regulatory elements, which can, e.g., affect the stability of the RNA in the eukaryotic cell, e.g., polyA tail. The RNA can encode one polypeptide. Alternatively, the RNA can be polycistronic and encode more than one polypeptide. The polypeptide can be, e.g., a vaccine antigen or an immunoregulatory molecule. The polypeptide can further be an endogenous or a foreign polypeptide. Foreign polypeptides include prokaryotic, e.g., bacterial, or viral polypeptides.
In another preferred embodiment, the RNA is an antisense RNA or a catalytic RNA, e.g., ribozyme. Preferred antisense RNAs or catalytic RNAs are capable of hybridizing to a nucleic acid in the eukaryotic cell, to thereby, e.g., regulate synthesis of a gene product.
In another embodiment, the invention provides an isolated bacterium comprising an RNA which is capable of being translated in a eukaryotic cell, or is an antisense RNA, or a catalytic RNA. In a preferred embodiment, the RNA is transcribed in the bacterium, e.g., from introduced DNA. In another embodiment, the RNA is introduced into the bacterium by, e.g., electroporation. The RNA can be heterologous with respect to the bacterium.
Preferred bacteria of the invention are invasive, i.e., capable of delivering at least one molecule, e.g., an RNA molecule, to a target cell, such as by invading the cytoplasm of the cell. Even more preferred bacteria are live bacteria, e.g., live invasive bacteria. More specifically, preferred bacteria of the invention are those capable of invading a vertebrate cell, e.g., a mammalian cell, such as a cell selected from the group consisting of a human, cattle, sheep, goat, horse, and primate cell.
A preferred invasive bacterium is Shigella, which is naturally invasive vis a vis vertebrate cells. At least one advantage of Shigella RNA vaccine vectors is their tropism for lymphoid tissue in the colonic mucosal surface. In addition, the primary site of Shigella replication is believed to be within dendritic cells and macrophages, which are commonly found at the basal lateral surface of M cells in mucosal lymphoid tissues. Thus, Shigella vectors provide a means to express antigens in these professional antigen presenting cells and thereby induce an immune response, e.g., a vaccine antigen.
Other naturally invasive bacteria include Listeria spp., Rickettsia spp. and enteroinvasive Escherichia coli. The term xe2x80x9cspp.xe2x80x9d refers to species of the genus preceding this term. In another embodiment, a bacterium can be modified, such as by genetic engineering means, to increase its invasive potential. In a preferred embodiment, the bacterium has been genetically engineered to mimic the invasion properties of Shigella spp., Listeria spp., Rickettsia spp. and enteroinvasive Escherichia coli. Any bacterium can be modified to increase its invasive potential and can be, e.g., a bacterium selected from the group consisting of Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus spp., Leishmania spp. and Erysipelothrix spp. In another embodiment, the bacterium is modified with an invasion factor, e.g., the bacterium is coated with an invasion factor, e.g., invasin.
The bacterium of the invention is preferably non-harmful to a subject to whom a bacterium of the invention is administered. Accordingly, the bacterium can be a naturally non-pathogenic bacterium. Alternatively, the bacterium can be an attenuated bacterium.
The invention further provides a pharmaceutical composition comprising any of the above-described bacteria and a pharmaceutically acceptable carrier, e.g., a physiological buffer and a lipoprotectant. Such pharmaceutical compositions can be used, e.g., for vaccinating an individual. Also within the scope of the invention are eukaryotic cells, e.g., human cells, comprising any of the above-described bacteria.
The invention also provides isolated DNA operably linked to a prokaryotic promoter, wherein the DNA encodes RNA which is capable of being translated in a eukaryotic cell, or is an antisense RNA or a catalytic RNA, e.g., ribozyme. Preferred prokaryotic promoters are the E. coli llp promoter and NirB promoter.
The invention further provides a method for introducing RNA into a eukaryotic cell. According to the invention, a eukaryotic cell is contacted with at least one invasive bacterium comprising a DNA molecule which is either capable of being transcribed into RNA in the bacterium or comprises RNA, wherein the RNA is capable of being translated in a eukaryotic cell or is an antisense RNA or catalytic RNA. The step of contacting the eukaryotic cell with at least one invasive bacterium can be performed in vitro at a multiplicity of infection ranging from about 0.1 to about 106 bacteria per eukaryotic cell. The contacting step is preferably performed in vitro at a multiplicity of infection from about 102 to about 104 bacteria per eukaryotic cell. In one embodiment, the contacting step is performed in vitro, and can, e.g., further comprise the step of administering the eukaryotic cell to a subject. In another preferred embodiment, the contacting step is performed in vivo, and comprises, e.g., administering to a subject the at least one bacterium, but preferably no more than about 1011 bacteria of the invention. In a preferred embodiment, from about 105 to about 109 bacteria are administered to a subject. The bacteria can be administered, e.g., orally, intrarectally, or intranasally to the subject. The bacterium can also be administered parenterally.
The invasive bacterium can be cell type specific. Alternatively, the invasive bacterium can be capable of invading one or more cell types. The invasive bacterium can also be modified to change its target specificity, e.g., by genetic engineering and/or by linking a specific targeting factor to the bacterium. In yet another embodiment, a non-invasive bacterium is be modified to become invasive. The bacterium can also be modified by engineering the bacterium to contain a suicide gene.
The eukaryotic cell to which the bacterium of the invention is targeted can be any type of cell. A preferred cell is from a mucosal tissue. In one embodiment, the cell is a natural target of the bacterium. In another embodiment, the target cell is modified, e.g., genetically, to contain a surface receptor necessary for mediating the interaction between the bacterium and the target cell.
Thus, the method of the invention retains all the advantages and properties of introducing RNA into a eukaryotic cell and provides a more efficient manner to deliver the RNA to the target eukaryotic cell. The advantages of introducing RNA into a eukaryotic cell instead of a DNA molecule include (i) avoidance of risk of insertion of DNA into the genome of the target eukaryotic cell and thus strongly reduced risk of mutation of the target eukaryotic cell; (ii) absence of need for the nucleic acid introduced in the eukaryotic cell to traverse the nuclear membrane; and (iii) avoidance of the possibility of shedding of plasmid molecules from the bacteria. Delivery of RNA to eukaryotic cells by use of a bacterium, compared to delivery of xe2x80x9cnakedxe2x80x9d RNA, e.g., where expression of the RNA is desired, provides at least the advantage that the RNA is protected and less likely to be degraded prior to entering the eukaryotic cell. Furthermore, the RNA can be specifically targeted to certain types of cells, since the bacterium can naturally target or be modified to target specific types of cells, e.g., antigen presenting cells in the mucosal lymphoid tissue.
Furthermore, the invention provides methods and compositions for oral vaccines, in particular, for an oral mucosal HIV-1 vaccine. Historically, oral vaccines have proven to be an efficacious means to invoke mucosal immunity. The invention provides oral vaccines using Shigella bacteria, which possess specialized adaptations that allow this organism to invade and replicate in the cytoplasm of antigen presenting cells associated with the colonic lymphoid tissue, thus eliciting strong immune responses. Thus, the invention provides efficacious oral vaccines.
Other features and advantages of the invention will be apparent from the following detailed description and claims.